Disruption of the mammalian Rad51 protein and disruption of proteins that associate with mammalian Rad51 for hindering cell proliferation

ABSTRACT

When a mutation, designated rad51 M1 , was generated in the mouse MmRAD51 gene, mutant embryos died shortly after implantation. rad51 M1  cells exhibited hypersensitivity to ionizing radiation, reduced proliferation, programmed cell death and chromosome loss. The disruption of MmRad51 protein--protein interactions stopped cell proliferation and/or reduced cell viability. Several proteins that interact with MmRad51 have been identified including, for example Brca2 and M96. Additionally, Rad51 self-associates via the N-terminal region. When a single residue was changed from a conserved lysine to an alanine, the alteration proved toxic to cells. Moreover, a rad51 allele that lacked the RecA homology region was also deleterious to cells. In view of the above, it is clear that inhibiting MmRad51 function or the function of any molecule that associates with MmRad51, or any molecule in the Rad51 or Rad52 pathways, hinders cell proliferation and/or viability. Accordingly, molecules capable of blocking these critical DNA repair pathways may be effective as therapeutics for inhibiting cell proliferation.

The present application is a continuation-in-part of and claims priorityto U.S. applications Ser. Nos. 08/758,280, filed Nov. 5, 1996. Thedisclosure of the above application is herein incorporated by reference.

1.0. FIELD OF THE INVENTION

The present invention relates to molecules that disrupt mammalian Rad51or Rad52 function, or disrupt the function of other molecules that areinvolved in the Rad51 or Rad52 pathways. Such molecules are useful as ameans to hinder cell proliferation or to promote programmed cell death,and define a novel class of therapeutic agents for use in the treatmentof proliferative disorders such as autoimmune disease and cancer.

2.0. BACKGROUND OF THE INVENTION

DNA repair and recombination are required by organisms to prevent theaccumulation of mutations and to maintain the integrity of geneticinformation. Compromised genetic material may result in cell cyclearrest, programmed cell death, chromosome loss or cell senescence.Alternatively, compromised genetic information may result indysregulation of the cell cycle ultimately leading to increased cellulargrowth and tumor formation.

The repair of double-strand breaks (DSB) in DNA is an essential cellularprocess. DSB repair may occur during general cellular functions such asDNA repair (Friedberg et al., 1995, DNA Repair and Mutagenesis. AmericanSociety for Microbiology, Washington, D.C.). In bacteria and yeastcells, DSB are predominately repaired by a homologous recombinationpathway (Krasin and Hutchinson, 1977, J. Mol. Biol. 116:81-98; Mortimer,1958, Radiat. Res. 9:312-16. In the budding yeast Saccharomycescerevisiae the RAD52 epistasis group (Rad₅₀ to Rad57, Mre11 and Xrs2)was identified in cells sensitive to ionizing radiation (reviewed inFriedberg, 1995; Petes et al., 1991, Recombination in yeast., p.407-521. In J. R. P. J. R. Broach, and E. W. Jones (ed.), The Molecularand Cellular Biology of the Yeast Saccharomyces. Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York). Later, some of themembers of this group were shown to be important for recombinationalrepair (e.g., Rad51, Rad52, Rad54, Rad55, Rad57 (Malkova et al., 1996,Proc. Natl. Acad. Sci. USA 93:7131-36, Sugawara et al., 1995, Nature373:84-86).

Among the members of the RAD52 epistasis group, ScRad51 is particularlyinteresting because it shares similarity with the Escherichia colirecombination protein, RecA. ScRad51 and RecA polymerize ondouble-stranded and single-stranded DNA (dsDNA, ssDNA) to produce ahelical filament, and both enzymes catalyze an ATP-dependent strandexchange between homologous DNA molecules (Ogawa et al., 1993, Science259:1896-99; Sung, 1994, Science 265:1241-4364; Sung and Robberson,1995, Cell 82:453-61). ScRad51 and RecA share 30% homology over a spanof about 220 amino acids, and each protein contains two conserved ATPbinding motifs (Aboussekhra et al., 1992, Mol. and Cell. Biol.12:3224-34; Basile et al., 1992, Mol. Cell. Biol. 12:3235-46; Sugawaraet al., 1995, Nature 373:84-86).

ScRad51 repairs DSB by homologous recombination. DSB accumulate atrecombination hot spots during meiosis in cells that lack ScRad51(Sugawara, 1995), and ScRad51 localizes to meiotic nuclei (Bishop, 1994,Cell 79:1081-92) and promotes meiotic chromosome synapsis (Rockmill etal., 1995, Genes & Develop. 9:2684-95). Accordingly, it is thought thatScRad51 mediates meiotic recombination by binding to single-strandsgenerated at DSB which are in strand pairing and exchange during meiosis(Sung and Robberson, 1995, Cell 82:453-61).

Direct and indirect protein--protein interactions are essential for RecAand ScRad51 function. The crystal structure of RecA suggests that aportion of the N-terminal region is involved in polymer formation (Storyet al., 1993, Science 259:1892-96; Story et al., 1992, Nature355:318-324) which was supported by genetic analysis that showedC-terminal truncations dominantly interfered with DNA repair inwild-type bacteria (Horii et al., 1992, J. Mol. Biol. 223:104-114;Tateishi et al., 1992, J. Mol. Biol. 223:115-129; Yarranton et al.,1982, Mol. Gen. Genet. 185:99-104). A similar self-association regionoccurs in the N-terminal region of ScRad51 and is essential for DNArepair (Donovan et al., 1994, Genes & Develop. 8:2552-2562; Shinohara etal., 1992, Cell 69:457-70). ScRad51 also associates with Rad52 and Rad55(Hays et al., 1995, Proc. Natl. Acad. Sci USA 92:6925-6929; Johnson andSymington, 1995, Molec. Cell. Biol. 15(9):4843-4850; Milne and Weaver,1993, Genes & Develop. 7:1755-1765) as well as other proteins. Otherprotein interactions may be inferred because a rad51 rad52A strain of S.cerevisiae was only partially complemented by Rad51 and Rad52 fromKluyveromyeces lactis (Donovan et al., 1994, Genes & Develop.8:2552-2562), and because ScRad51 colocalized with Dmc1 to thesynaptonemal complex (Bishop, 1994, Cell 79:1081-92). These data suggestthat a large protein complex is necessary for recombinational repair andthat disruption of any of the proteins in this complex hinders therepair of DSB.

RecA/ScRad51 homologues have been discovered in a wide range oforganisms including the fission yeast Schizosaccharomyces pombe (Jang etal., 1994, Gene 142:207-11; Muris et al., 1993, Nuc. Acids Res.21:4586-91; Shinohara et al., 1993, Nature Genet. 4:239-4358), lilies(Terasawa et al., 1995, Genes & Develop. 9:925-34), chickens (Bezzubovaet al., 1993, Nucl. Acids Res. 21:1577-80), mice (Morita et al., 1993,Proc. Natl. Acad. Sci USA 90:6577-80; Shinohara et al. 1993, NatureGenet. 4:239-43) and humans (Shinohara et al. 1993; Yoshimura et al.,1993, Nucl. Acids Res. 21:1665), and appear to be involved in DNA repairand recombination based on the following evidence: 1) Conserved RecAhomology--MmRad51 is 83% homologous, 69% identical to ScRad51, and 51%homologous, 28% identical to RecA. Shared homology between mammalian andyeast Rad51 suggest conserved function due to the remarkable similaritybetween other mammalian and yeast DNA repair pathways (reviewed inCleaver, 1994, Cell 76:1-4); 2) Expression pattern--MmRAD51 is highlyexpressed in tissues involved in meiotic recombination such as testes(Morita et al., 1993, Proc. Natl. Acad. Sci USA 90:6577-80) and ovaries(Shinohara et al., 1993, Nature Genet. 4:239-43). Additionally,expression of the S. pombe MmRad51 homologue SpRAD51 increased aftercells were treated with methyl methanesulfonate which provides furtherevidence of a DNA repair function (Jang et al., 1994, Gene 142:207-11);3) Protein cellular localization--Mouse, chicken, and lily Rad51localizes at discrete foci on meiotic chromosomes at varyingconcentrations during prophase 1, possibly on the lateral elements andrecombination nodules, which suggests a role in the repair of DSB duringmeiotic recombination (Ashley et al., 1995, Chromosoma 104:19-28; Haafet al., 1995, Proc. Natl. Acad. Sci. USA 92:2298-2302; Terasawa et al.,1995). Moreover, increasing concentrations of human Rad51, HsRad51,localize to the nucleus after exposure to DNA damaging agents which alsosuggests a repair function (Terasawa et al., 1995); 4) Filamentformation on DNA--HsRad51 bind to ssDNA which demonstrates a potentialfor strand exchange (Benson et al., 1994, EMBO 13:5764-71); 5) Mousecells with a rad51 mutation, designated rad51^(M1), displayed featuresthat are known to be characteristic of unrepaired DSB in yeast cells(Lim and Hasty, 1996, In press) which include reduced proliferation,hypersensitivity to γ-radiation, chromosome loss and programmed celldeath.

3.0. SUMMARY OF THE INVENTION

An object of the present invention is to hinder cell proliferation orreduce cell viability by disrupting mammalian Rad51 function.

An additional object is to hinder cell proliferation or reduce cellviability by disrupting mammalian Rad52 function.

Another object of the present invention is to hinder cell proliferationor reduce cell viability by disrupting proteins that associate withmammalian Rad51.

Another object of the present invention is to hinder cell proliferationor reduce cell viability by disrupting proteins that associate withmammalian Rad52.

Another object of the present invention is to hinder cell proliferationor reduce cell viability by disrupting any proteins involved in themammalian Rad51 or mammalian Rad52 pathways.

Another object of the present invention is to hinder cell proliferationor reduce cell viability by disrupting mammalian Rad₅₁ proteininteractions.

Another object of the present invention is to hinder cell proliferationor reduce cell viability by disrupting mammalian Rad52 proteininteractions.

Another object of the present invention is to hinder cell proliferationor reduce cell viability by disrupting protein--protein interactionsthat are involved in the mammalian Rad51 or mammalian Rad52 pathways.

Yet another embodiment of the present invention involves methods ofidentifying compounds that are capable of inhibiting the binding orfunction of any protein involved in the Rad51 pathway, and, inparticular, compounds capable of binding or inhibiting the function ofRad51 protein. Accordingly, an additional embodiment of the presentinvention involves methods of screening for compounds that disruptdouble-stranded break repair by assaying for microsatellite formation incells; assaying for chromosome loss in cells; assaying for thedisruption of strand exchange in an in vitro assay; assaying fordecreased cell proliferation; assaying for premature replicativecellular senescence; and assaying for increased cell death.

Another object of the invention is to identify compounds capable ofinterfering with protein--protein interactions involved in DSB repair byscreening large numbers of compounds in assays that allow the detectionof a decrease in protein--protein interactions. In a further object ofthe invention, structural analysis of proteins, peptides, and compoundsuseful for modulating DSB repair is used to improve the modulation ofDSB repair by new or known proteins, peptides, and compounds.

An additional object of the present invention are compounds that hindercell proliferation or reduce cell viability by disrupting mammalianRad51 function.

An additional object of the present invention are compounds that hindercell proliferation or reduce cell viability by disrupting mammalianRad52 function.

4.0. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. mRNA structure of MmRAD51. The predicted amino acids arenumbered according to Shinohara et al., 1993. The shaded box representsthe recA homology region. The open boxes represent regions that are notconserved across species. The thick vertical lines represent the ATPbinding domains.

FIG. 2. MmRad51 self-association as demonstrated by the yeast two-hybridsystem. The self-association is restricted to the most N-terminal 43amino acids. The shaded box is the RecA core homology region (Shinoharaet al, 1993). The thick vertical lines represent the ATP-binding sites.The open boxes represent regions that are not conserved between species.The relative β-galactosidase (β-gal) activities are presented, rightpanel. Full length wild-type MmRad51 is considered to be 100%. E12served as a negative control and had 1% relative activity.

FIG. 3. Targeting the transgenes to the hprt locus. The hprt sequencescontain exons 2 and 3 (labeled boxes). Hprt homology of vector origin isa thick line, of chromosomal origin is a thin line. The bacterialplasmid is represented by a wavy line. Potential locations forcrossovers are X's labeled 1 or 2. Two recombination events arepossible: either gene replacement with crossovers at both 1 and 2 orvector insertion with crossovers at either 1 or 2. For vector insertion,only a crossover at 1 is shown.

FIGS. 4A and 4B. Disruption of mammalian Rad51 function in cells. A)Conditional expression of mammalian Rad51 1-43 in ES cells increasessensitivity to gamma-radiation. Transgene turned on without Dox andturned off with Dox. Ten clones were observed and the averages arepresented. B) Brca2 peptide decreases proliferation of p53--/--fibroblasts. 50 micromolar concentration used. Colony size is based oncell number. The average of two experiments is shown when 100 cells wereplated onto a 6 cm plate. The control is either no peptide or the 16amino acid peptide derived from Antennapedia.

4.0. DETAILED DESCRIPTION OF THE INVENTION

As discussed above, one embodiment of the present invention is theexpression of altered mammalian rad51 alleles that disrupt mammalianRad51 function, mammalian Rad52 function, or the function of any otherprotein in the mammalian Rad51 or Rad52 pathways. The function ofMmRad51 is not entirely known; however, it is likely that it has thesame function as ScRad51 which is recombinational repair. Therecombinational repair pathway appears to be at least partiallyconserved between yeast and mammals. Mammalian homologues were found formembers of the Rad52 epistasis group (Rad51, Rad52) and to other yeastproteins (Dmc1) implicated in recombinational repair (Malkova et al.,1996, Proc. Natl. Acad. Sci. USA 93:7131-36; Resnick et al., 1989, Proc.Natl. Acad. Sci. USA 86:2276-80; Tsuzuki et al., 1996, Proc. Natl. Acad.Sci USA 93:6236-40). Expression pattern supported the hypothesis thatthese homologues maintained the same function from yeast to mammals.MmRAD51 was highly expressed in tissues with cells involved in meioticrecombination, testis and ovary, and rapid cell division, intestine,embryo, and thymus (Morita et al., 1993; Shinohara et al., 1993). A roleduring meiotic recombination was further suggested because MmRAD51 washighly enriched in the synaptonemal complex in pachytene spermatocytes(Ashley et al., 1995; Haaf et al., 1995).

The most compelling evidence that MmRad51 and ScRad51 function isconserved comes from analysis of rad51 mutant cells, the mutation wasdesignated rad51^(M1) (Lim and Hasty, 1996). rad51^(M1) cells exhibitedreduced proliferation, hypersensitivity to γ-radiation, chromosome lossand cell death. These characteristics were similar to yeast cellsdeficient for recombinational repair either due to sequence divergenceor due to a mutation in rad51 or rad52. Even though these data suggestMmRad51 functions during recombinational repair it is possible that thesevere phenotype in rad51^(M1) cells was due to disruption of anotherprocess.

There is evidence that the RecA homologues perform multiple tasks, someof them not shared by the others. Two RecA homologues were found inMyxococcus xanthus, only one was essential, but both complemented UVsensitivity in an E. coli recA strain (Norioka et al., 1995, J.Bacteriol. 177:4179-82). Two RecA homologues found in yeast, ScRad51 andDmc1, are essential for meiotic recombination, but only ScRad51 isessential for mitotic recombination (Bishop, 1994, Rockmill et al. 1995,Genes & Develop. 9:2684-95). In mammals, a Dmc1 homologue was isolatedsuggesting that RecA homologues possess diverse and unique functions inmammalian cells (Habu et al., 1996).

Reduced proliferation, hypersensitivity to γ-radiation, chromosome loss,and cell death have all been associated with rad51^(M1) cells. Thesecharacteristics are similar to those seen in yeast cells deficient forrecombinational repair either due to sequence divergence, or due to amutation in rad51 or rad52 (Malkova et al., 1996, Proc. Natl. Acad. Sci.USA 93:7131-36; Resnick et al., 1989, Proc. Natl. Acad. Sci. USA86:2276-80; Tsuzuki et al., 1996, Proc. Natl. Acad. Sci USA 93:6236-40).Even though these data suggest MmRad51 functions during recombinationalrepair it is also possible that the severe phenotype observed inrad51^(M1) cells was due to disruption of another process.

For the purposes of the present application the term ionizing radiationshall mean all forms of radiation, including but not limited to a, P,and T radiation and U.V. light, which are capable of directly orindirectly damaging the genetic material of a cell or virus. The termirradiation shall mean the exposure of a sample of interest to ionizingradiation, and the term radiosensitive shall refer to cells orindividuals which display unusually adverse consequences after receivingmoderate, or medically acceptable (i.e., nonlethal diagnostic ortherapeutic doses), exposure to ionizing irradiation.

MmRad51 may perform a novel role in DNA replication, repair, orchromosomal disjunction. MmRAD51 expression is restricted during thecell cycle to late G₁ /S/G₂ and MmRAD51 expression was activated bymitogens that induced T and B cell proliferation suggesting a role inreplication and repair (Yamamoto et al., 1996, 251:1-12). MmRad51 maytake part in disjunction because it localizes to the kinetochores ofdiakinesis, and metaphase 1 chromosomes (Ashley et al., 1995).

The exact function or functions performed by MmRad51 are unimportantwith regard to developing anti-proliferative drugs and cancertherapeutics as long as the disruption of the MmRad51 function providesa benefit to the patient. For the purposes of the present invention, itis assumed that the function of Rad51 is the repair of DSB; however, itis likely that Rad51 performs additional functions in the cell. However,it is important to note that at least some aspect of MmRad51 function isessential for cell proliferation and/or viability, and that moleculescapable of disrupting MmRad51 function thus hinder cell proliferation orreduce cell viability. As such, any molecule that disrupts the MmRad51pathway should prove useful for cancer therapy (for example).

Furthermore, disruption of any protein--protein interaction thatinvolves either MmRad51 or any other molecule in the MmRad51 pathwayshould also prove useful for cancer therapy.

Protein--protein interactions are critical for recombinational repair inyeast cells, including interactions that involve ScRad51 and ScRad52(Donovan et al., 1994; Milne et al., 1993). In addition, the human Rad51and Rad52 proteins were shown to associate like their yeast homologues(Shen et al., 1996, J. Biol. Chem. 271:148-152).

To isolate proteins that associate with MmRad51, a yeast two-hybridscreen was performed with MmRad51 as the "bait" and a T cell library andan embryonic cell library as the "prey". Among other proteins identifiedusing this screen, MmRad51 and Brca2 were isolated, and the interactionsidentified using this screen may prove critical for in vivo function.Additional biochemical binding assays that may prove useful foridentifying compounds that are able to associate with MmRad51 (or anyother target protein) are well known in the art including, but notlimited to: equilibrium or membrane flow dialysis, antibody bindingassays, gel-shift assays, in vitro binding assays, filter bindingassays, enzyme-linked immunoabsorbent assays (ELISA), western blots,co-immunoprecipitation, immunogold co-immunoprecipitation,coimmunolocalization, co-crystallization, fluorescence energy transfer,competition binding assays, chemical crosslinking, and affinitypurification. In addition, genetic analysis may be used to identifyaccessory proteins that interact with MmRad51 or are peripherallyinvolved in MmRad51 function. Where the MmRad51 accessory protein isessential to MmRad51 function, mutation in the genes encoding theseproteins should typically result in phenotypes similar to thoseassociated with MmRad51 mutations. Similarly, where the MmRad51accessory proteins function to inhibit or retard MmRad51 activity,mutations in the genes encoding these factors shall generally mimicantagonist phenotypes.

The MmRad51 self-association was investigated further. Deletion analysisrevealed that the MmRad51 self-association occurred in the N-terminalregion which further demonstrated conservation of function with ScRad51and RecA since both were shown to self-associate via the N-terminalregion of the protein (Donovan et al., 1994; Horii, 1992; Story et al.,1992, 1993; Tateishi et al., 1992; Yarranton and Sedgwick, 1982).Although the presently described invention has been specificallyexemplified using a species exemplary of the order mammalia, given therelatively high level of interspecies sequence similarity (andfunctional similarity) observed in the Rad51 proteins, it is clear thatthe present invention may be broadly applied to other mammalian species,including humans, as well as non-mammalian animals such as birds, andfish.

In addition to mice, examples of mammalian species that may be used inthe practice of the present invention include, but are not limited to:humans, non-human primates (such as chimpanzees), pigs, rats (or otherrodents), rabbits, cattle, goats, sheep, and guinea pigs.

Given the critical importance of mammalian Rad51 function, anydisruption of the mammalian Rad51 or Rad52 complexes, or any member intheir pathway will necessarily hinder cell proliferation or viability.When the Rad51 and Rad52 pathways were disrupted by introducing alteredmouse rad51 into mouse cells, nonproductive protein--proteinassociations resulted. The altered forms of mouse rad51 were generatedby disrupting a conserved nucleotide binding motif while preserving theprotein association domain. The expression of these transgenes resultedin cellular toxicity. Presumably, the resulting nonproductive proteinassociations were responsible for the drastically reduced viability ofthese cells. In view of this result, it is clear one may reduce cellproliferation by disrupting mammalian Rad51 function, or the function ofany protein in this repair pathway by hindering protein association byusing defective proteins or other means such as small molecules.

Given that the Rad51 proteins are known to self-associate, the Rad51protein sequence provides a template for the identification and genesisof peptides or factors that disrupt Rad51 function or activity For thepurposes of the present invention a "peptide" is any sequence of atleast about five amino acids up to about 100 amino acids. Typically, thepeptides of the present invention can encompass enzymatic domains, DNA,RNA, or protein binding domains, or any fragment of a protein or aminoacid sequence that directly or indirectly provide the desired functionof disrupting cellular Rad51 or Rad52 activity. Accordingly, anadditional embodiment of the present invention are peptides orpolypeptides that correspond at least five contiguous amino acids of themammalian Rad51 amino acid sequence (SEQ ID NO. 1), or the human Rad51amino acid sequence (SEQ ID NO. 2) that retain the property of beingcapable of binding a mammalian Rad51 and/or inhibiting Rad51 function(as detected using a suitable biochemical, genetic, or cellular assay).

Additionally, the blocking of normal Rad51 function may induceprogrammed cell death. Thus, one aspect of the present invention are anovel class of therapeutic agents, factors, or compounds that have beenengineered, or are otherwise capable of disrupting the essentialprocesses that are mediated by, or associated with, normal Rad51 orRad52 activity. Accordingly, it is contemplated that this novel class oftherapeutics agents may be used to treat diseases including, but notlimited to, autoimmune disorders and diseases, inflammation, cancer,graft rejection, and any of a variety of proliferative orhyperproliferative disorders.

Typical examples of therapeutic agents based on the above presentlydescribed molecules include, but are not limited to, defective (eitherengineered or naturally occurring) forms of the proteins that associatewith the protein complexes, inhibitory fragments of the proteins, wildtype and altered genes that code for proteins that disrupt mammalianRad51 function, small organic molecules, antisense nucleic acidsequences, oligonucleotides that inhibit expression or activity via atriplex mechanism, peptides, aptameric oligonucleotides, and the like.

More particularly, examples of engineered proteins may include, but arenot limited to, proteins that comprise inactivating mutations inconserved active sites (e.g., ATP binding motifs, DNA or protein bindingdomains, catalytic sites, etc.), fusion proteins that comprise at leastone inhibitory domain, and the like.

The above agents may be obtained from a wide variety of sources. Forexample, standard methods of organic synthesis may be used to generatesmall organic molecules that mimic the desired regions of the target DNArepair proteins. In addition, combinatorial libraries comprising a vastnumber of compounds (organic, peptide, or nucleic acid, reviewed inGallop et al. 1994, J. Med. Chem. 37(9):1233-1251; Gordon et al., 1994,J. Med. Chem. 37(10):1385-1401; and U.S. Pat. No. 5,424,186 all of whichare herein incorporated by reference) may be screened for the ability tobind and inhibit the activity of proteins involved in DSB repair or anyother potential mammalian Rad51 function.

In particular, inhibitory peptides should prove very useful. Suchcompounds may include, but are not limited to, peptides such as, forexample, soluble peptides, including but not limited to members ofrandom peptide libraries; (see, e.g., Lam et al., 1991, Nature354:82-84; Houghten et al., 1991, Nature 354:84-86), and combinatorialchemistry-derived molecular library made of D- and/or L- configurationamino acids, phosphopeptides (including, but not limited to members ofrandom or partially degenerate, directed phosphopeptide libraries; see,e.g., Songyang et al., 1993, Cell 72:767-778).

Given that an important aspect of DSB repair is the interaction ofproteins, additional aspects of the invention are the use of screeningassays to detect interactions or the lack of such interactions ofproteins involved in DSB repair. The following assays are designed toidentify compounds that interact with (e.g., bind to) proteins involvedin DSB repair. The compounds which may be screened in accordance withthe invention include but are not limited to peptides, antibodies andfragments thereof, prostaglandins, lipids and other organic compounds(e.g., terpines, peptidomimetics) that bind to or mimic the activitytriggered by a natural ligand (i.e., agonists) or inhibit the activitytriggered by a natural ligand (i.e., antagonists) of a protein involvedin DSB repair; as well as peptides, antibodies or fragments thereof, andother organic compounds that mimic the natural ligand for a givenprotein involved in DSB repair.

Such compounds may include, but are not limited to, peptides such as,for example, soluble peptides, including but not limited to members ofrandom peptide libraries (see, e.g., Lam, K. S. et al., 1991, Nature,354:82-84; Houghten, R. et al., 1991, Nature, 354:84-86), andcombinatorial chemistry-derived molecular library peptides made of D-and/or L- configuration amino acids, phosphopeptides (including, but notlimited to members of random or partially degenerate, directedphosphopeptide libraries; see, e.g., Songyang, Z. et al., 1993, Cell,72:767-778); antibodies (including, but not limited to, polyclonal,monoclonal, humanized, anti-idiotypic, chimeric or single chain=antibodies, and FAb, F(ab)₂ and FAb expression library fragments, andepitope-binding fragments thereof); and small organic or inorganicmolecules.

Other compounds which can be screened in accordance with the inventioninclude but are not limited to small organic molecules that are able togain entry into an appropriate cell and affect DSB repair by, forexample, modulating protein--protein interactions important for DSBrepair (e.g., by interacting with a protein involved in DSB repair); orsuch compounds that affect the activity of a gene encoding a proteininvolved in DSB repair.

Computer modeling and searching technologies permit identification ofcompounds, or the improvement of already identified compounds, that canmodulate DSB repair by, for example, modulating protein--proteininteractions involved in DSB repair. Having identified such a compoundor composition, the active sites or regions are identified. Such activesites might typically be the binding partner sites, such as, forexample, the interaction domains of a protein important for DSB repairwith its cognate ligand. The active site can be identified using methodsknown in the art including, for example, from the amino acid sequencesof peptides, from the nucleotide sequences of nucleic acids, or fromstudy of complexes of the relevant compound or composition with itsnatural ligand. In the latter case, chemical or X-ray crystallographicmethods can be used to find the active site by finding where on thefactor the complexed ligand is found.

Next, the three dimensional geometric structure of the active site isdetermined. This can be done by known methods, including X-raycrystallography, which can determine a complete molecular structure. Onthe other hand, solid or liquid phase NMR can be used to determinecertain intra-molecular distances. Any other experimental method ofstructure determination can be used to obtain partial or completegeometric structures. The geometric structures may be measured with acomplexed ligand, natural or artificial, which may increase the accuracyof the active site structure determined.

If an incomplete or insufficiently accurate structure is determined, themethods of computer based numerical modeling can be used to complete thestructure or improve its accuracy. Any recognized modeling method may beused, including parameterized models specific to particular biopolymerssuch as proteins or nucleic acids, molecular dynamics models based oncomputing molecular motions, statistical mechanics models based onthermal ensembles, or combined models. For most types of models,standard molecular force fields, representing the forces betweenconstituent atoms and groups, are necessary, and can be selected fromforce fields known in physical chemistry. The incomplete or lessaccurate experimental structures can serve as constraints on thecomplete and more accurate structures computed by these modelingmethods.

Finally, having determined the structure of the active site, eitherexperimentally, by modeling, or by a combination thereof, candidatemodulating compounds can be identified by searching databases containingcompounds along with information on their molecular structure. Such asearch seeks compounds having structures that match the determinedactive site structure and that interact with the groups defining theactive site. Such a search can be manual, but is preferably computerassisted. The compounds found from such a search generally identifymodulating compounds, or genes encoding the same, that are selected forfurther study or gene targeting.

Alternatively, these methods can be used to identify improved modulatingcompounds from an already known modulating compound or ligand. Thecomposition of the known compound can be modified and the structuraleffects of modification can be determined using the experimental andcomputer modeling methods described above applied to the newcomposition. The altered structure is then compared to the active sitestructure of the compound to determine if an improved fit or interactionresults. In this manner systematic variations in composition, such as byvarying side groups, can be quickly evaluated to obtain modifiedmodulating compounds or ligands of improved specificity or activity.

Further experimental and computer modeling methods useful to identifymodulating compounds based upon identification of the active sites ofregulatory protein interactions, and related transduction factors willbe apparent to those of skill in the art.

Representative examples of molecular modeling systems include the CHARMmand QUANTA programs (Polygen Corporation, Waltham, Mass.). CHARMmperforms the energy minimization and molecular dynamics functions.QUANTA performs the construction, graphic modeling and analysis ofmolecular structure. QUANTA allows interactive construction,modification, visualization, and analysis of the behavior of moleculeswith each other.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds whichare inhibitors or activators of the proteins and genes that areimportant for any aspect of DSB repair.

In vitro systems may be designed to identify compounds capable ofinteracting with (e.g., binding to) the regulatory proteins identifiedusing the subject methods. The identified compounds may be useful, forexample, in modulating the activity of wild type and/or mutant proteinsimportant for DSB repair. In vitro systems may also be utilized toscreen for compounds that disrupt normal interactions important for DSBrepair.

The assays used to identify compounds that bind to proteins importantfor DSB repair involve preparing a reaction mixture of a given proteinand the test compound under conditions and for a time sufficient toallow the two components to interact and bind, thus forming a complexwhich can be removed and/or detected in the reaction mixture. Theprotein used can vary depending upon the goal of the screening assay.For example, where agonists of the natural ligand are sought, a fulllength protein, or a fusion protein containing a protein or polypeptidethat affords advantages in the assay system (e.g., labeling, isolationof the resulting complex, etc.) can be utilized.

The screening assays can be conducted in a variety of ways. For example,one method to conduct such an assay would involve anchoring the protein,polypeptide, peptide or fusion protein or the test substance onto asolid phase and detecting binding between the protein and test compound.In one embodiment of such a method, the protein reactant may be anchoredonto a solid surface, and the test compound, which is not anchored, maybe labeled, either directly or indirectly. In another embodiment of themethod, the test protein is anchored on the solid phase and is complexedwith a labeled antibody (and where a monoclonal antibody is used, it ispreferably specific for a given region of the protein). Then, a testcompound could be assayed for its ability to disrupt the association ofthe protein/antibody complex.

In practice, microtiter plates, or any modernized iteration thereof, mayconveniently be utilized as the solid phase. The anchored component maybe immobilized by non-covalent or covalent attachments. Non-covalentattachment may be accomplished by simply coating the solid surface witha solution of the protein and drying. Alternatively, an immobilizedantibody, preferably a monoclonal antibody, specific for the protein tobe immobilized may be used to anchor the protein to the solid surface.The surfaces may be prepared in advance and stored.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynonimmobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously nonimmobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the previously nonimmobilizedcomponent (the antibody, in turn, may be directly labeled or indirectlylabeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for the testprotein, polypeptide, peptide or fusion protein, or the test compound toanchor any complexes formed in solution, and a labeled antibody specificfor the other component of the possible complex to detect anchoredcomplexes.

Macromolecules that interact with a given protein important for DSBrepair are referred to, for purposes of this discussion, as "bindingpartners". Therefore, it is desirable to identify compounds thatinterfere with or disrupt the interaction with such binding partnerswhich may be useful in modulating DSB repair.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between a protein and its binding partneror partners involves preparing a reaction mixture containing the testprotein, polypeptide, peptide or fusion protein as described above, andthe binding partner under conditions and for a time sufficient to allowthe two to interact and bind, thus forming a complex. In order to test acompound for inhibitory activity, the reaction mixture is prepared inthe presence and absence of the test compound. The test compound may beinitially included in the reaction mixture, or may be added at a timesubsequent to the addition of the test protein and its binding partner.Control reaction mixtures are incubated without the test compound orwith a placebo.

The formation of any complexes between the test protein and the bindingpartner is then detected. The formation of a complex in the controlreaction, but not in the reaction mixture containing the test compound,indicates that the compound interferes with the interaction of the testprotein and the binding partner.

The assay for compounds that interfere with protein binding can beconducted in a heterogeneous or homogeneous format. Heterogeneous assaysinvolve anchoring either the test protein or the binding partner onto asolid phase and detecting complexes anchored on the solid phase at theend of the reaction. In homogeneous assays, the entire reaction iscarried out in a liquid phase. The examples below describe similarassays which may be easily modified to screen for compounds whichdisrupt or enhance the interaction. In either approach, the order ofaddition of reactants can be varied to obtain different informationabout the compounds being tested. For example, test compounds thatinterfere with the interaction by competition can be identified byconducting the reaction in the presence of the test substance; i.e., byadding the test substance to the reaction mixture prior to orsimultaneously with the test protein and interactive binding partner.Alternatively, test compounds that disrupt preformed complexes, e.g.,compounds with higher binding constants that displace one of thecomponents from the complex, can be tested by adding the test compoundto the reaction mixture after complexes have been formed. The variousformats are described briefly below.

In a heterogeneous assay system, either the test protein, or theinteractive binding partner, is anchored onto a solid surface, while thenon-anchored species is labeled, either directly or indirectly. Inpractice, microtiter plates are conveniently utilized. The anchoredspecies may be immobilized by non-covalent or covalent attachments.Non-covalent attachment may be accomplished simply by coating the solidsurface with a solution of the test protein or binding partner anddrying. Alternatively, an immobilized antibody specific for the speciesto be anchored may be used to anchor the species to the solid surface.The surfaces may be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, may bedirectly labeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the test protein and theinteractive binding partner is prepared in which either protein islabeled, but the signal generated by the label is quenched due toformation of the complex (see, e.g., U.S. Pat. No. 4,109,496 byRubenstein which utilizes this approach for immunoassays). The additionof a test substance that competes with and displaces one of the speciesfrom the preformed complex will result in the generation of a signalabove background. In this way, test substances which disrupt the bindinginteraction can be identified.

For an example of a typical labeling procedure, a test protein or apeptide fragment, e.g., corresponding to the relevant binding domain,can be fused to a glutathione-S-transferase (GST) gene using a fusionvector, such as pGEX-5X-1, in such a manner that its binding activity ismaintained in the resulting fusion protein. The interactive bindingpartner can be labeled with radioactive isotope, for example, by methodsroutinely practiced in the art. In a heterogeneous assay, e.g., theGST-fusion protein can be anchored to glutathione-agarose beads. Theinteractive binding partner can then be added in the presence or absenceof the test compound in a manner that allows interaction and binding tooccur. At the end of the reaction period, unbound material can be washedaway. The interaction between the fusion product and the labeledinteractive binding partner can be detected by measuring the amount ofradioactivity that remains associated with the glutathione-agarosebeads. The successful inhibition of binding by the test compound willresult in a decrease in measured radioactivity.

Alternatively, the GST-fusion protein and the labeled interactivebinding partner can be mixed together in liquid in the absence of thesolid glutathione-agarose beads. The test compound can be added eitherduring or after the species are allowed to interact. This mixture canthen be added to the glutathione-agarose beads and unbound material iswashed away. Again the extent of binding inhibition can be measured bydetermining the amount of radioactivity associated with the beads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the test proteins, in place of the full length proteins. Any numberof methods routinely practiced in the art can be used to identify andisolate the binding sites. These methods include, but are not limitedto, mutagenesis of the gene encoding the protein and screening fordisruption of binding in a co-immunoprecipitation assay. Sequenceanalysis of the gene encoding the protein will reveal the mutations thatcorrespond to the region of the protein involved in interactive binding.

The invention encompasses cell-based and animal model-based assays forthe identification of compounds exhibiting the ability to alter orcorrect phenotypes associated with the various genotypes identified andconstructed using the present methods. Such cell-based assays can alsobe used as the standard to assay for purity and potency of thecompounds, including recombinantly or synthetically produced proteins orcompounds.

Given that they will serve as templates for the rational design ofagents for disrupting DSB repair activity in the cell, it would beadvantageous to purify each of the individual proteins that are directlyor indirectly involved in DSB repair of any other potential mammalianRad51 function. The various proteins involved in the DSB repair pathwaysmay be purified using any of a number of variations of well establishedbiochemical, and molecular biology techniques. Such techniques are wellknown to those of ordinary skill in the biochemical arts and have beenextensively described in references such as Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology, Volume 152,Academic Press, San Diego, Calif. (1987; Molecular Cloning: A LaboratoryManual, 2d ed., Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989);Current Protocols in Molecular Biology, John Wiley & Sons, all Vols.,1989, and periodic updates thereof); New Protein Techniques: Methods inMolecular Biology, Walker, J. M., ed., Humana Press, Clifton, N.J.,1988; and Protein Purification: Principles and Practice, 3rd. Ed.,Scopes, R. K., Springer-Verlag, New York, N.Y., 1987. In general,techniques including, but not limited to, ammonium sulfateprecipitation; centrifugation, ion exchange, gel filtration, andreverse-phase chromatography (and the HPLC or FPLC forms thereof) may beused to purify the various proteins of the DSB repair complex.

Additionally, purified preparations of the presently described DNArepair proteins, associated proteins, or fragments thereof, may be usedto generate antisera specific for a given agent. Accordingly, additionalembodiments of the present invention include polyclonal and monoclonalantibodies that recognize epitopes of the presently described DNA repaircomplex proteins. The factors used to induce the antibodies of interestneed not be biologically active; however, the factors should induceimmunological activity in the animal used to generate the antibodies.

Given that similar methodologies may be applied to the generation ofantibodies to the various factors, for purposes of convenience, only theRad51 factor antibodies will be discussed further.

Polypeptides for use in the induction of Rad51-specific antibodies mayhave an amino acid sequence consisting of at least three amino acids,and preferably at least 10 amino acids, that mimic a portion of theamino acid sequence of Rad51, and may contain the entire amino acidsequence of naturally occurring Rad51 or a Rad51-derivative.

Anti-Rad51 antibodies are expected to have a variety of medically usefulapplications, several of which are described generally below. Moredetailed and specific descriptions of various uses for anti-Rad51antibodies are provided in the sections and subsections which follow.Briefly, anti-Rad51 antibodies may be used for the detection andquantification of Rad51 polypeptide expression in cultured cells, tissuesamples, and in vivo. Such immunological detection of Rad51 may be used,for example, to identify, monitor, and assist in the prognosis ofneoplasms that have been treated with factors that inhibit DSB repair.Additionally, monoclonal antibodies recognizing epitopes from differentparts of the Rad51 structure may be used to detect and/or distinguishbetween native Rad51 and various subcomponent and/or mutant forms of themolecule. Additionally, anti-Rad51 monoclonal antibodies may be used totest preparations of agents or factors that mimic segments of Rad51, orare designed to impair protein association with Rad51, or tocompetitively inhibit DNA binding. In addition to the various diagnosticand therapeutic utilities of anti-Rad51 antibodies, a number ofindustrial and research applications will be obvious to those skilled inthe art, including, for example, the use of anti-Rad51 antibodies asaffinity reagents for the isolation of Rad5l-associated polypeptides,and as immunological probes for elucidating the biosynthesis, metabolismand biological functions of Rad51. Rad51 antibodies may also be used topurify Rad51 or Rad51-associated factors by affinity chromatography.

Once purified, the proteins of interest may be partially sequenced, andthese data may be used to design degenerate oligonucleotide probes foruse in cloning the genes encoding the various proteins that areassociated with DSB repair. Alternatively, any of a variety of public orprivate sequence data bases may be searched for nucleic acid or peptidesequences that share homology with genes and proteins associated withRad51-mediated DSB repair. Once a similar sequence is identified,peptides may be produced and screened for inhibitory activity. Where anucleic acid library is involved, one could synthesize a probecorresponding to the nucleic acid sequence of interest, and use theprobe to clone a full-length version of the corresponding gene (ifnecessary). Accordingly, an additional embodiment of the presentlyclaimed invention are nucleic acid sequences that are capable ofhybridizing to sequences encoding the proteins that are associated withDSB repair under stringent conditions. For the purposes of the presentinvention, the term "stringent conditions" generally refers tohybridization conditions that (1) employ low ionic strength and hightemperature for washing, for example, 0.015 M NaCl/0.0015 M sodiumcitrate/0.1% SDS at 50° C.; or (2) employ during hybridization adenaturing agent such as formamide, for example, 50% (vol/vol) formamidewith 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C.; or (3) employ 50 formamide, 5× SSC (0.75 M NaCl,0.075 M Sodium pyrophosphate, 5× Denhardt's solution, sonicated salmonsperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., withwashes at 42° C. in 0.2× SSC and 0.1% SDS. The above examples ofhybridization conditions are merely provided for purposes ofexemplification and not limitation. A more thorough treatise of the suchroutine molecular biology techniques may be found in Sambrook et al.,Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, New York, Vols. 1-3: (1989), and periodic updates thereof,herein incorporated by reference.

Once isolated, the genes encoding the proteins involved in DSB repairmay be recombinantly expressed using standard vectors and hosts.Examples of vectors that may be used to express proteins of interest areprovided in Sambrook et al., Molecular Cloning, Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York, Vols. 1-3: (1989). Inparticular, eucaryotic viruses may be used as vectors to transduce anyof a wide variety of plant and animal cells to overexpress the desiredproteins. Examples of such viruses include, but are not limited to,adenovirus, papilloma virus, herpes virus, adeno-associated virus,rabies virus, bacculo virus, retrovirus, plant viruses, and the like(See generally, Sambrook et al., Molecular Cloning, Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York, Vol. 3:16.1-16.89(1989); U.S. Pat. No. 5,316,931, issued May 31, 1994, hereinincorporated by reference).

Preferably, agents that disrupt DSB repair shall be substantiallyspecific for blocking the desired repair pathways. For the purposes ofthe present invention, the term substantially specific shall mean that agiven agent is capable of being dosaged to provide the desired effectwhile not causing undue cellular toxicity.

One of ordinary skill will appreciate that, from a medicalpractitioner's or patient's perspective, virtually any alleviation orprevention of an undesirable symptom (e.g., symptoms related to disease,sensitivity to environmental factors, normal aging, and the like) wouldbe desirable. Thus, for the purposes of this Application, the terms"treatment", "therapeutic use", or "medicinal use", used herein shallrefer to any and all uses of compositions comprising the claimed agentswhich remedy a disease state or symptoms, or otherwise prevent, hinder,retard, or reverse the progression of disease or other undesirablesymptoms in any way whatsoever.

When used in the therapeutic treatment of disease, an appropriate dosageof presently described agents, or derivatives thereof, may be determinedby any of several well established methodologies. For instance, animalstudies are commonly used to determine the maximal tolerable dose, orMTD, of bioactive agent per kilogram weight. In general, at least one ofthe animal species tested is mammalian. Those skilled in the artregularly extrapolate doses for efficacy and avoiding toxicity to otherspecies, including human. Before human studies of efficacy areundertaken, Phase I clinical studies in normal subjects help establishsafe doses.

Additionally, the bioactive agents may be complexed with a variety ofwell established compounds or structures that, for instance, enhance thestability of the bioactive agent, or otherwise enhance itspharmacological properties (e.g., increase in vivo half-life, reducetoxicity, etc.).

Another aspect of the present invention includes formulations thatprovide for the sustained release of DSB repair antagonists. Examples ofsuch sustained release formulations include composites of biocompatiblepolymers, such as poly(lactic acid), poly(lactic-co-glycolic acid),methylcellulose, hyaluronic acid, collagen, and the like. The structure,selection and use of degradable polymers in drug delivery vehicles havebeen reviewed in several publications, including, A. Domb et al.,Polymers for Advanced Technologies 3:279-292 (1992). Additional guidancein selecting and using polymers in pharmaceutical formulations can befound in the text by M. Chasin and R. Langer (eds.), "BiodegradablePolymers as Drug Delivery Systems, " Vol. 45 of "Drugs and thePharmaceutical Sciences," M. Dekker, New York, 1990. Liposomes may alsobe used to provide for the sustained release of DSB repair antagonists.Details concerning how to use and make liposomal formulations of drugsof interest can be found in, among other places, U.S. Pat. No 4,944,948;U.S. Pat. No. 5,008,050; U.S. Pat. No. 4,921,706; U.S. Pat. No.4,927,637; U.S. Pat. No. 4,452,747; U.S. Pat. No. 4,016,100; U.S. Pat.No. 4,311,712; U.S. Pat. No. 4,370,349; U.S. Pat. No. 4,372,949; U.S.Pat. No. 4,529,561; U.S. Pat. No. 5,009,956; U.S. Pat. No. 4,725,442;U.S. Pat. No. 4,737,323; U.S. Pat. No. 4,920,016. Sustained releaseformulations are of particular interest when it is desirable to providea high local concentration of DSB repair antagonist, e.g., near a tumor,site of inflammation, etc.

Where diagnostic, therapeutic or medicinal use of the presentlydescribed agents, or derivatives thereof, is contemplated, the bioactiveagents may be introduced in vivo by any of a number of establishedmethods. For instance, the agent may be administered by inhalation; bysubcutaneous (sub-q); intravenous (I.V.), intraperitoneal (I.P.), orintramuscular (I.M.) injection; or as a topically applied agent(transdermal patch, ointments, creams, salves, eye drops, and the like).

Additionally, an alternative means for employing the presently disclosedanti-proliferation agents includes the use of vectors to directly insertgenes encoding the agents into target cells (e.g., gene therapy). Forexample, when the tumor cells express the genes encoding the desiredsequences, DSB repair will be disrupted and the tumor cell will die.Alternatively, one could attack tumor cells using a strategyconceptually similar to that disclosed in U.S. Pat. No. 5,529,774 hereinincorporated by reference. In brief, cells that produce transducingvirus encoding sequence that disrupts DSB repair may be implanted at ornear the tumor mass. As the producer cells continue to elaborate virus,the growing tumor cells are infected and effectively killed as theyexpress the agent that blocks DSB repair. The above methodology hasproven useful in the treatment of glioblastomas and other tumors of thebrain by using retroviral vectors to selectively target activelyreplicating tumor cells. A similar methodology could be used to deliverantisense sequences that target (and thus inhibit) the expression ofRad51 or any of the proteins involved in the Rad51 or Rad52 pathways.

The mammalian Rad₅₁ or Rad52-mediated repair pathways, and theassociated proteins, are essential for cell proliferation or viability.These DNA repair pathways most likely function by repairing DSB viahomologous recombination between sister chromatids during S/G₂(recombinational repair); however, during G₁, the repair of DSB may alsooccur via nonhomologous recombination (nonhomologous end joining). Thenonhomologous recombination pathway was once thought to be the majorrepair pathway in mammalian cells. Much of this belief stems from genetargeting data that demonstrated homologous recombination to be lessfrequent than random or illegitimate recombination (Bradley et al.,1992, Bio/Technology 10:534-39). Other data demonstrated thatchromosomal DSB frequently were joined without homology or with onlyvery short stretches of homology (Rouet and Jasin, 1994, Mol. Cell.Biol. 14:8096-8105). DNA-dependent protein kinase (DNA-PK) is criticalfor nonhomologous but not homologous repair of DSB (Liang et al., 1996,Proc. Natl. Acad. Sci. USA 93:8929-33). A biphasic response to ionizingradiation was observed in DNA-PK-deficient cell lines with resistance inlate S phase suggesting that DNA-PK functions in G₁ and another repairpathway functions in S phase (Jeggo, 1990, Mutation Research 239:1-16).DNA-PK is composed of a catalytic subunit called DNA-PK_(cs) and a DNAend-binding subunit called Ku which is a heterodimer of Ku70 and Ku86(Park et al., 1996, J. Biol. Chem. 1996:18996-19000, for review, seeRoth et al., 1995; Shen et al., 1996. Analysis 30 of DNA-PK activity hascome from scid (severe combined immunodeficient) mice which aredeficient in DNA-PK_(cs) (Kirchgessner et al., 1995, Science267:1178-82), and Ku86-deficient mice (Nussenzweig et al., 1996, Nature382:551-55; Zhu et al., 1996, Cell 86:379-89). Both scid andKu86-deficient mice are immune deficient due to a defect in repair ofDSB generated during V(D)J recombination. Unfortunately, it isimpossible to analyze V(D)J recombination in rad51-mutant mice or cells;however, it is unlikely that MmRad51 plays a role in this process sinceMmRad51 localizes to the nucleus in late G₁ through G₂ (Yamamoto et al.,1996, 251:1-12), and V(D)J recombination occurs in G₀ /G₁ (Schlissel etal., 1993, Genes & Dev. 7:2520-32). In general, scid and Ku86-deficientcells do have similarities to MmRad51-deficient cells. All arehypersensitive to ionizing radiation, and Ku86-deficient cells wereprematurely senescent in tissue culture, indicating a similar function.However, since scid and Ku86-deficient mice and cells were viable andMmRad51-deficient cells were not, the consequences of removing theputative homologous recombination pathway to repair DSB appears to bemore vital than the removal of the nonhomologous pathway.

The presently described DSB repair antagonists are particularly deemeduseful for the treatment of cancer. Cancers that may be treated by themethods of the invention include, but are not limited to: Cardiac:sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma),myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogeniccarcinoma (squamous cell, undifferentiated small cell, undifferentiatedlarge cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastom,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tutrr, chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma [pinealoma], glioblastoma multiforme, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cord(neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma, [serous cystadenocarcinoma,mucinous cystadenocarcinoma, endometrioid tumors, celioblastoma, clearcell carcinoma, unclassified carcinoma], granulosa-thecal cell tumors,Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva(squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma,fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cellcarcinoma, botryoid sarcoma [embryonal rhabdomyosarcoma], fallopiantubes (carcinoma); Hematoloqic: blood (myeloid leukemia [acute andchronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia,myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignantlymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Karposi's sarcoma, moles, dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma.

In addition to cancer, the presently disclosed compounds are effectiveagainst any of a wide variety of hyperproliferative disorders including,but not limited to: autoimmune disease, arthritis, inflammatory boweldisease, proliferation induced after medical procedures, including, butnot limited to, surgery, angioplasty, and the like.

The anti-cancer application of agents that functionally disruptmammalian Rad51, Rad52 or any member in the DSB repair pathway, requiresthat DSB repair remains equally critical in cancer cells. Cancer cellslack many of the normal cell cycle regulatory mechanisms that arecritical to controlling proliferation, and inducing programmed celldeath, and it remains possible that the absence of these mechanismsrenders Rad51 and/or Rad52 function nonessential. The protein p53 iscentral to regulation of the cell cycle, and stimulation of cell deathin response to DNA damage including DNA damaged by ionizing radiation(reviewed by Ko and Prives, 1996, Genes & Develop. 10:1054-72). p53 isthe most commonly mutated gene in cancer cells (Donehower et al., 1992,Nature 356:215-21; Vogelstein, 1990, Nature 348:681-682) and mutationsin p53 are known to increase cell proliferation and promote chromosomalinstability (Harvey et al., 1993, Oncogene 8:2457-67).

The early lethal phenotype in rad51^(M1) mutant embryos and cells may bestimulated by a cell cycle response to unrepaired DNA damage. DNA damagewas shown to inhibit progression through the cell cycle, demonstrating arelationship between DNA lesions and cell cycle proteins (Carr andHoekstra, 1995, Trends in Cell Biology 5:32-40). In mitotically dividingbudding yeast cells, a single DSB in a dispensable plasmid wassufficient to induce cell death, partly under the control of Rad9(Bennett et al., 1993, Proc. Natl. Acad. Sci. USA 90:5613-17; Schiest1et al., 1989, Mol. Cell. Biol. 9:1882-9654, Weinert and Hartwell, 1988,Science 241:317-22). In mammalian cells, the tumor suppressor gene, p53,responded to DNA damage induced by γ-radiation by delaying the cellcycle, or inducing programmed cell death (Kastan et al., 1991, CancerResearch 51:6304-11; Kuerbitz et al., 1992, Proc. Natl. Acad. Sci. USA89:7491-95). These responses may be the critical tumor suppressorfunction of p53 (Baker et al., 1990, Science 249:912-15; Lowe et al.,1994, Science 266:807-10, Symonds et al., 1994, Cell 78:703-11).Induction of p53 after exposure to ionizing radiation and restrictionendonuclease suggest that the formation of DSB may initiate a p53response (Lu and Lane, 1993, Cell 75:765-78).

p53 was at least partly responsible for regulating the rad51^(M1)phenotype because development was extended from the early egg cylinderstage to the head fold stage in a p53-mutant background. However, thedouble-mutant embryos died from either accumulation of DNA damageresulting in metabolic incompetence and mitotic failure, orp53-independent regulation. Murine embryonic fibroblasts, generated fromdouble-mutant embryos, failed to proliferate and were completelysenescent in tissue culture; thus, demonstrating that MmRad51 functionwas critical in cells that exhibit chromosomal instability andaccelerated proliferation. It is therefore likely that disruption ofMmRad5 or any other protein in its pathway or disruption of anyprotein--protein interaction important in the DSB repair pathway resultsin reduced proliferation or decreased cell viability. This featureremains true even in cells with reduced capacity to regulate the cellcycle.

The present invention is further illustrated by the following examples,which are not intended to be limiting in any way whatsoever.

5.0. EXAMPLES

5.1. Cloning of the Mouse MmRAD51 cDNA

The MmRAD51 cDNA sequence was cloned and used to generate an expressionvector. The 5' end of cDNA was amplified by RT-PCR from mouse testis RNAand was then used as a probe to screen a mouse brain cDNA library. Oneclone was identified and sequenced. The coding sequence was identical toMmRAD51 disclosed in published reports (Morita et al., 1993; Shinoharaet al., 1993); however, the clone contained about 300 additional basepairs of 5' noncoding sequence and about 400 extra base pairs of 3'noncoding sequence (FIG. 1).

5.2. The Use of a Yeast Two-Hybrid Screen to Isolate Proteins ThatAssociate with MmRad51

ScRad51 was shown to self-associate as well as associate with otherproteins such as ScRad52 and ScRad55 (Donovan et al., 1994; Hays et al.,1995; Johnson and Symington, 1995; Milne and Weaver, 1993; Shinohara etal., 1992). Kluyveromyeces lactis RAD51 and RAD52 did not rescue arad51Δ rad52Δ strain of S. cerevisiae and overexpression of ScRAD51suppressed rad55 and rad57 mutant yeast which indicates interactingproteins are necessary (Donovan et al., 1994; Hays et al., 1995). Also,Dmc1 and ScRad51 colocalized to the synaptonemal complex which suggestedthat they act together during meiotic recombination (Bishop, 1994).

The modified yeast two-hybrid system was used to isolate proteins thatassociate with mammalian Rad51 which is a genetic screen for determiningprotein--protein interactions (Harper et al., 1993). One of the proteinsis a hybrid of the GAL4 DNA-binding domain fused to MmRad51 (the"bait"). The other is a hybrid of the GAL4 transactivating domain fusedto an embryonic or a T cell cDNA library (the "prey"). The bait and preywere co-expressed in HF7c yeast that contained two reporters, HIS3 andlacZ fused to the GAL4 promoter and grown in media lacking histidine andcontaining 25 mM 3-AT (an antimetabolite;3-amino-1,2,4-triazole)-Functional GAL4 was created when the DNA bindingdomain and the transactivation domain were juxtaposed, ideally by aMmRad51-protein interaction. Such an interaction induced the HIS3 andlacZ genes allowing a positive colony to survive in medium lackinghistidine and to turn blue in X-Gal(5-bromo-4-chloro-3-indolyl-β-D-galactosidase).

Seven specific clones were isolated from this screen. A 13.5 dayembryonic cDNA library (500 μg) was transfected into 5×10⁶ cells andplated onto forty 15 cm plates. A T cell cDNA library (400 μg) wastransfected into 4×10⁶ cells and plated onto twenty 15 cm plates. Atotal of 80 His⁺ colonies grew in about 3 days. Of these, 40 turned blueafter about 5 to 30 minutes of exposure to X-gal. These colonies weretested for specificity by transfecting HF7c cells without bait or with anonspecific bait (E12). Nonspecific associations were observed in 20clones. The inserts in the other clones were sequenced and 13 were outof frame and seven were in frame. The sequences for the remaining sevenclones were screened in the GCG data base. Homologues were found forfour clones and three clones were novel (Table 1). The protein producedby clone 1 was 100% homologous to MmRad51 which showed that the screenwas successful because RecA and ScRad51 are both known toself-associate. The protein produced from clone 2 was 100% homologous toa metal response element binding protein, M96 (Inouye et al., 1994, DNAand Cell Biol. 13(7):731-742). The function of M96 is unknown. Theprotein produced from clone 3 was 48% homologous to human XP-G (ERCC-5)and 45% homologous to chicken Histone Hi. A mutation in XP-G isresponsible for the genetic disorder xeroderma pigmentosum (Cleaver,1994; Cleaver and Kraemer, 1995, In The metabolic basis of inheriteddisease, p. 4393-4419, 7th ed. McGraw-Hill, New York.). XP-G is ahomologue of the S. cerevisiae excision repair protein, ScRad2 which isa ssDNA endonuclease. It is possible that MmRad51 repairs single-strandbreaks as well as double-strand breaks and that single-strand breaks caninitiate recombination. Histone H1 is a component of the nucleosome andcomprises a group of related proteins that vary in tissues and arepoorly conserved across species. The length of DNA may be affected byHistone Hi binding to the linker region and joining adjacentnucleosomes. The protein produced from clone 4 was 100% homologous tothe human breast cancer gene, BRCA2 (Tavtigian et al., 1996, Nat. Gen.12:333-337; Wooster et al., 1995, Nature 378:789-792). The function ofBrca2 is unknown; however, like p53, it is a tumor suppressor gene andmay therefore regulate the cell cycle in response to DNA damage. Thus,the observed association with a DNA repair gene, MmRad51, is consistentwith such an activity.

                  TABLE 1                                                         ______________________________________                                        Clones isolated from the yeast two-hybrid screen                                Clone      Homology             Library                                     ______________________________________                                        1        100% to MmRad51      T cell                                            2 100% to M96 embryo                                                          3 45% to Histone H1, 48% to XP-G embryo                                       4 100% to Brca2 T cell                                                        5 novel T cell                                                                6 novel embryo                                                                7 novel embryo                                                              ______________________________________                                    

Clones isolated from a yeast two-hybrid screen with MmRad51 as the"bait" and an embryonic or T cell cDNA library as the "prey". Theinserts obtained from the prey were sequenced and compared to sequencesin the GCG data base. The measured extent of protein homology is listed.All clones strongly associated with MmRad51 in the N-terminal region(amino acids 1-43). Colonies grew within three days in 3-AT, and cellsgenerally stained blue after about 5 minutes of X-gal exposure.

5.3. Deletion Analysis of MmRad5 to Isolate the Protein AssociationRegion

A deletion analysis was performed to isolate the MmRad51self-association domain. Full length MmRAD51 was used as the bait anddeletions of 51RAD51 were the prey (FIG. 2). The "prey" MmRad51deletions were individually co-transfected with the bait into HF7ccells. The relative levels of β-galactosidase activity were measured forthe MmRad51 deletion proteins as compared to full length MmRad51 whichwas considered to have 100% activity. Expression of the C-terminalregion, TR43-339 and TR131-339 did not result in blue yeast cells after10 hours, and the relative β-galactosidase activity was about 1, or thesame as for the nonspecific bait, E12. However, expression of theN-terminal region, TR1-43, stained yeast cells blue in less than 5minutes and the relative β-galactosidase activity was 43%.Interestingly, a sequence containing more of the N-terminal region ofthe protein, TR1-93, caused the yeast cells to stain blue after about 30minutes of X-gal exposure, and reduced the relative β-galactosidaseactivity to about 4%. In similar experiments, TR1-131 and TR1-175respectively displayed 11% and 9% of the β-galactosidase activity of thepositive control. Nevertheless, these data indicated that the N-terminalregion was responsible for MmRad51 self-association. It also appearedthat amino acids 43-93 inhibited self-association and that thisinhibition was relieved by adding more of the C-terminal region of theprotein. These data indicated that MmRad51 was functionally conservedwith ScRad51 since the self-association domain was also in theN-terminal region for both proteins even though these regions did notdisplay conserved amino acid sequences.

The other six proteins listed in Table 1 were tested to determine ifthey interacted with the N-terminal region of MmRad51. All six stronglyinteracted with TR1-43; thus, the most N-terminal 43 amino acids wereresponsible for all the MmRad51 protein--protein interactions observed.Given the high level of homology shared between the human and murineRad51 proteins (in the important N-terminal self-association region, theproteins only differ at amino acid positions 10 and 46 where the humansequence respectively contains an asparagine in lieu of the serine, anda phenylalanine in place of the tyrosine encoded by the mouseprotein--both relatively conservative replacements), the presentlydescribed results should reflect the results expected from similarstudies using the human Rad51 protein.

5.4. Transfection of Mouse Embryonic Stem Cells with Altered Alleles ofMammalian rad51

Both MmRad51 and ScRad51 self-associate using their respectiveN-terminal regions. This observation supports the hypothesis that theseproteins remain functionally conserved. Functional conservation wasfurther tested in the RecA core homology domain. In ScRad51, the RecAcore homology region was shown to be essential for the repair of DSB.The gene rad51K-A191 was altered in the first ATP-binding motif, and aconserved Lysine was changed to an Alanine. The expression ofrad51K-A191 in wild-type yeast cells dominantly impaired the repair ofDNA damage and generated a rad51 null phenotype. Nonproductiveprotein--protein interactions were probably responsible for the dominantnegative phenotype because rad51K-A191 was shown to associate withwild-type ScRad51 and ScRad52. If the MmRad51 structural domains weresimilar to ScRad51, then disruption of the conserved Lysine in the firstATP-binding motif should result in a null phenotype because of thenonfunctional associations with wild-type MmRad51 or other proteins inthis pathway such as mouse Rad52 or Brca2. A null rad51 mutationresulted in a severe cell proliferation defect that preventedpropagation of mutant mouse cells in tissue culture. Therefore, cellsthat expressed a dominant negative rad51 allele should not be recovereddue to this proliferation defect.

Altered alleles of mammalian rad51 that were engineered to be dominantnegative were expressed in mouse embryonic stem cells. Due to theseverity of the null phenotype, these experiments were designed tomeasure the absence of transfected cells by statistically relevantnumbers. The first experiment measured the transfection efficiencies ofvectors that expressed altered mammalian rad51 as compared to a vectorthat expressed wild-type mammalian RAD51, or vector alone. The alteredtransgenes, rad51TR1-131 and rad51K-A134, contained a functional proteinbinding region and a nonfunctional RecA homology region. Forrad51TR1-131, a C-terminal truncation was made in the first ATP-bindingdomain (FIG. 1). For rad51K-A134, the conserved Lysine in the firstATP-binding motif was changed to an Alanine (for review, see Donovan andWeaver, 1994). rad51K-A134 more strongly associated with full lengthMmRad51 than rad51 TR1-131 as measured using the yeast two-hybrid systemwith about 90% relative β-galactosidase activity (FIG. 1). The alteredand wild-type transgenes were cloned into a CMV expression vector with aneomycin phosphotransferase (neo) cassette (pcDNA3 from invitrogen).Transfected embryonic stem (ES) cells were selected in G418 and colonieswere counted 9 days later. The altered transgenes generated 20-30% fewerG418^(r) colonies as compared to colonies resulting after transfectionwith wild-type MmRAD51 or vector alone in three experiments. Variationsof 20-30% in transfection frequencies are commonly observed and areconsequently not determinative in and of themselves. However, thisminimal reduction could also indicate that the toxic product of thealtered transgenes was produced in sufficient quantities to stop cellproliferation. However, if the transgene product was truly toxic, thenwhy did 70-80% of the cells survive in selection media? The transgenemay be silent while the neo gene is expressed. The transgene may bedisrupted upon integration into the chromosome or by chromosomalpositional effects. In addition, strong expression of the transgene maybe required to observe a phenotype while only weak expression of neo maybe required for positive selection. Another experiment was needed tocircumvent these possible problems.

5.5. Targeting the Expression Vectors to the RPRT Locus

Another experiment was developed to compare the targeting frequencies ofvectors that expressed altered mammalian rad51 with vectors thatexpressed wild-type mammalian RAD51 or MC1tk (Herpes Simplex Virus type1 thymidine kinase). The transgenes were targeted to the hypoxanthinephosphoribosyltransferase locus, HPRT (Melton et al., 1984, Proc. Natl.Acad. Sci. USA 81:2147-2151). Targeting the transgenes to HPRT woulddecrease the likelihood of disruption upon integration and Southernanalysis could also be used to verify the integrity of the integrationevent (FIG. 3). The transgenes would also be located to a favorableenvironment for expression since HPRT is a house keeping gene, and thusall of the transgenes would be affected to the same degree by chromatinpositional effects. The transgenes were cloned into the bacterialplasmid of an insertion vector that targeted HPRT (IVH). There were 6.9kb of HPRT sequences that contained a neo cassette in exon 3. Therefore,upon linearization using a unique site in the homology region (anengineered NotI site), both insertion and replacement events could berecovered.

The targeting vectors were linearized in the HPRT homology region andtransfected into ES cells. Transfected cells were selected for by growthin medium containing G418, and targeted cells were selected in mediumcontaining G418+6-thioguanine (TG). G418 resistant (G418^(r) ) colonieswere counted to measure the transfection efficiency and TG^(r) +G418^(r)colonies were counted to measure the targeting frequency.

                  TABLE 2                                                         ______________________________________                                        Targeting frequencies                                                                                                  target                                  No.    frequency                                                              of total total TG.sup.r + G418.sup.r relative                                Exp. Exps. G418.sup.r TG.sup.r G418.sup.r to IVH-tk                         ______________________________________                                        IVH-tk  A      2      4088  338  1/12    NA                                     IVH-51TA  1  636 34 1/19 -37%                                                 IVH-51KA  2 2792 106 1/26 -54%                                                IVH-tk B 2 1200 124 1/10 NA                                                   IVH-51TA  2  472 22 1/21 -52%                                                 IVH-51KA  2 1504 62 1/24 -58%                                                 IVH-tk C 2 6016 264 1/23 NA                                                   IVH-51WT  2 4840 192 1/26 -12%                                                IVH-51TA  2 2584 70 1/37 -38%                                                 IVH-51KA  2 3664 48 1/76 -70%                                                 IVH-tk D 2 6744 414 1/16 NA                                                   IVH-51KA  2 4848 136 1/37 -57%                                                IVH-tk E 2 2624 186 1/14 NA                                                   IVH-51WT  2 1456 84 1/17 -18%                                                 IVH-51TA  2 2208 96 1/23 -39%                                                 IVH-51KA  2 1376 52 1/26 -46%                                                 IVH-tk F 2 1664 156 1/11 NA                                                   IVH-51WT  2  752 60 1/12  -8%                                                 IVH-51TA  2  760 34 1/22 -50%                                                 IVH-51KA  2  544 30 1/18 -39%                                               ______________________________________                                    

Table 2: Electroporation: 10 μg of NotI cut DNA/10⁷ cells/ ml PBS, 575V/cm and 500 μF. Each experiment (exps. A--F) shows results fromelectroporations that were done on the same day with a common batch ofES cells under identical conditions to eliminate variability. NA, notapplicable.

The targeting frequencies of vectors that contained altered rad51alleles were compared to control vectors (Table 2). Vectors thatcontained altered rad51 alleles were IVH-51TR1-131 (containsrad51TRI-131) and IVH-51KA (contains rad51K-A134). Control vectors wereIVH-51WT (contains wild-type MmRAD51), and IVH-tk (contains MC1tk). Therelative targeting frequencies (TG^(r) +G418^(r) /G418^(r) colonies)were determined using IVH-tk efficiency as 100%. The relative targetingfrequencies were reduced by 13+/-3.6% for IVH-51WT (average of threeexperiments), 43+/-6.4% for IVH-51TR1-131 (average of 5 experiments) and54+/-7.6% for IVH-51KA (average of six experiments).

Southern analysis was performed on TG^(r) +G418^(r) clones to verifytargeting and to identify the different targeting patterns (FIG. 3).Several types of recombination patterns were possible. A vectorinsertion event would integrate the entire vector to form a duplicationof HPRT homology (Hasty et al., 1992, Molec. and Cell. Biol.12:2464-2474). The vector may integrate on the 5' long arm or the 3'short arm (rarely observed). These integration patterns were combinedsince both integrate the transgene in between the duplication. A genereplacement event would introduce the neo but not the transgene andthus, provided a control. Modified events, that were not predicted byeither pattern could also occur, and an intact transgene may or may notbe introduced.

Comparison of the targeting patterns for the four vectors indicated thatthe transgene product was toxic for both rad51TR1-131 and rad51K-A134.The relative percentage of clones targeted with IVH-51TR1-131 andIVH-51KA that contained the transgene (vector insertion) decreased, andthe relative percentage of targeted clones that did not contain thetransgene (gene replacement) increased relative to controls. For bothIVH-tk and IVH-51WT, targeting usually occurred by vector insertion (75%and 80%, respectively), rarely by gene replacement (14% and 17%,respectively), or more rarely by a modified event (6% and 8%,respectively). However, for IVH-51TR1-131 and IVH-51KA the relativefrequency of targeted events that occurred by vector insertion decreased(68% and 45%, respectively), and gene replacement events increased (27%and 41%, respectively). The relative frequency of modified events alsoincreased for clones targeted with IVH-51KA (14%). Therefore, thealtered transgenes rarely integrated into the target locus as comparedto the controls.

5.6. A High Percentage of Transfected Clones did not Express theTransgene

A statistically significant reduction in targeting frequency wasobserved using vectors that contained the altered rad51 alleles ascompared to the wild-type allele or MC1tk. In addition, alteredtransgenes were introduced into HPRT for a lower percentage of thetargeted clones as compared to the controls. However, targeted cloneswere generated that appeared to incorporate the altered transgenesintact. There are several possibilities for survival: 1) A smallmutation may have been generated in the transgene; 2) The chromatinstructure of the transgene may have been altered during the targetingevent to silence the transgene (or vice- versa); 3) Position effectvariegation may inhibit transcription of the transgene, but not neo.

Expression of MC1tk was tested in clones targeted with IVH-tk todetermine the fraction of clones that do not express the transgene.Sixty-two TG^(r) +G418^(r) clones were grown in replica plates, onewithout FIAU and one with FIAU, to distinguish clones that lost ormaintained HSV-1 thymidine kinase activity. A large percentage of clones(42%) survived in FIAU demonstrating that the IVH-51TR1-131 and IVH-51KAtargeting frequencies were reduced to background levels. Therefore, allof the cells targeted with either IVH-51TR1-131 and IVH-51KA thatexpress the transgene were probably not recovered.

5.7. Conditional Expression of Amino Acids 1-43 of Mammalian Rad51 in EScells Increases Sensitivity to γ-radiation

It was demonstrated that the rad51^(M1) mutation increases sensitivityto γ-radiation in early E3.5 day embryos and that dominant negativetransgenes decrease proliferation of ES cells. Now, an expression vectorthat codes for amino acids 1-43 of Rad51 and is conditionally regulatedby Doxycycline ("Dox") was introduced into the Hprt locus of ES cells.The expression vector is turned off by 5 ng/ml Dox. A vector thatexpresses the inducible tetR gene was transfected into the ES cells withthe Rad51 1-43 expression vector in the presence of 5 ng/ml Dox. Tenclones were analyzed for sensitivity to γ-radiation when grown in mediawith or with out Dox. Cells grown with Dox (transgene turned off) weremore resistant to γ-radiation than cells grown without it, demonstratingthat amino acids 1-43 of Rad51 sensitizes cells to radiation (FIG. 4a).Since ES cells are immortal and transformed, these data demonstrate thatdisrupting the Rad51 pathway will serve as a therapeutic for cancer.Therefore, expression of dominant negative transgenes that code for anyprotein that will disrupt mammalian Rad51 function could serve as atherapeutic for cancer and should not be limited to just the first 43amino acids of Rad51.

5.8. Application of a Peptide that Inhibits Cell Proliferation byDisrupting Mammalian Rad51

It was demonstrated that mammalian Rad51 interacts with mammalian Brca2.A peptide of the amino acid sequenceROIKIWFONRRMKWKKFLSRLPLPSPVSPICTFVSPAAQKAFQPPRS was synthesized. Thispeptide contains the region of Brca2 that interacts with Rad51, aminoacids 3196-3226 (not underlined), SEQ ID NO. 3. This peptide alsocontains 16 amino acids derived from the Drosophila Antennapedia protein(underlined) SEQ ID NO:4that translocates through biological membranes.This peptide was added to media after p53^(-/-) fibroblasts were platedat low concentration (100 cells/ 6 cm plate). Colonies were countedbased on size as determined by the number of cells. The peptide caused agreat reduction of colonies composed of 265 or greater cells (FIG. 4b).Thus, the peptide had a profoundly negative effect on cellularproliferation. The 16 amino acids derived from Antennapedia had noeffect on the number of colonies at any size; therefore, the inhibitoryaffect was due to the Brca2 sequences. Since p53^(-/-) cells are highlyproliferative and commonly found in cancer, these data demonstrate thatdisrupting the Rad51 pathway will serve as a therapeutic for cancer.Therefore, any peptide that interacts with mammalian Rad⁵¹ may inhibitproliferation of cells in tissue culture and could be used to inhibitthe growth of cancer.

5.9. Application of Molecules that Disrupt Mammalian Rad51 and/or Rad52Function for Cancer Therapeutics

The rad51^(M1) mutation reduces proliferation and promotes cellularsenescence, even in a p53 mutant background. In addition, rad51 dominantnegative alleles also display this phenotype by presumably formingnonproductive protein associations with Rad51 and other proteins likeRad52, M96 and Brca2. Therefore, it is likely that the disruption ofmammalian Rad51, mammalian Rad52 (or any protein in the DSB repairpathway mediated by these proteins) will reduce cell proliferation orinduce cell death, and thus be suitable as a cancer therapeutic. Inaddition, the disruption of any protein--protein association importantfor mammalian Rad51 function or mammalian Rad52 function will alsoreduce cell proliferation or induce cell death, and thus be suitable asa cancer therapeutic.

Additionally, dominant negative alleles of rad51 may be used to expresscancer therapeutics that reduce cell proliferation or induce cell death.An expression vector that codes for a dominant negative rad51 allele maybe introduced into cancer cells, or an mRNA that codes for a dominantnegative rad51 allele may be introduced into cancer cells, or a dominantnegative Rad51 protein may be introduced into cancer cells. Severalexamples of such dominant negative rad51 alleles are presentlydisclosed. Of these alleles, the protein encoded by rad51K-A131 appearsto have the strongest self-association, and proved toxic toproliferating cells. In fact, any rad51 allele that rendered the RecAhomology region nonfunctional but preserved the N-terminal proteinassociation region should reduce cell proliferation or induce cell deathand could thus be used as a cancer therapeutic.

In addition to subtle alterations in the RecA core homology region ofmammalian Rad51, C-terminal truncations in mammalian rad51 may also beused to reduce cell proliferation and/or induce cell death. rad51TR1-131demonstrated a toxic effect on cells even though it had a relativelyweak interaction with MmRad51 which suggested that the phenotype mightbe caused by nonfunctional self-associations, or nonfunctionalassociations with other proteins such as Rad52, M96 and Brca2.rad51TR1-43 had a strong interaction with MmRad51 and may be moreeffective as a cancer therapeutic than rad51TR1-131. In fact, anyC-terminal truncation that preserves the protein interacting region ofRad51 may be used as a dominant negative allele for cancer therapy.Additionally, fusion of the N terminal domain of mammalian Rad51 to the16 or 60 amino acids of the 3rd helix of the antennapedia protein maypromote entry into the nucleus (Derossi et al., 1994, J. Bio. Chem.269:10444-10450).

Mammalian Rad51 interacts with other proteins besides itself, anddisruption of these interactions could be used to reduce cellproliferation or induce cell death. Other proteins interacting withmammalian Rad51 include but are not limited to mammalian Rad52, Brca2and M96.

The identification of other interacting proteins will further elucidatethe pathway and present greater opportunities to disrupt this pathwayfor the purpose of hindering cell proliferation. Since mammalian Rad52associates with mammalian Rad51 and other proteins (Park et al., 1996;Shen et al., 1996), dominant alleles of mammalian Rad52 may also hindercell proliferation or induce cell death. Such alleles could also be usedfor cancer therapeutics. In fact, dominant alleles of any protein thatassociates with mammalian Rad51, Rad52 or any other protein in thesepathways, may be expected to hinder cell proliferation or induce celldeath. Thus, all of the above molecules collectively define a new classof therapeutic agents for the treatment of proliferative disorders,viral infection (especially HIV infection), and cancer.

EQUIVALENTS

The foregoing specification is considered to be sufficient to enable oneskilled in the art to broadly practice the invention. Indeed, variousmodifications of the above-described methods for carrying out theinvention, which are obvious to those skilled in the field ofmicrobiology, biochemistry, organic chemistry, medicine or relatedfields, are intended to be within the scope of the following claims. Allpatents, patents applications, and publications cited herein areincorporated by reference.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 4                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 339 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Met Ala Met Gln Met Gln Leu Glu Ala Ser Al - #a Asp Thr Ser Val        Glu                                                                              1               5  - #                10  - #                15              - - Glu Glu Ser Phe Gly Pro Gln Pro Ile Ser Ar - #g Leu Glu Gln Cys Gly                  20      - #            25      - #            30                   - - Ile Asn Ala Asn Asp Val Lys Lys Leu Glu Gl - #u Ala Gly Tyr His Thr              35          - #        40          - #        45                       - - Val Glu Ala Val Ala Tyr Ala Pro Lys Lys Gl - #u Leu Ile Asn Ile Lys          50              - #    55              - #    60                           - - Gly Ile Ser Glu Ala Lys Ala Asp Lys Ile Le - #u Thr Glu Ala Ala Lys      65                  - #70                  - #75                  - #80        - - Leu Val Pro Met Gly Phe Thr Thr Ala Thr Gl - #u Phe His Gln Arg Arg                      85  - #                90  - #                95               - - Ser Glu Ile Ile Gln Ile Thr Thr Gly Ser Ly - #s Glu Leu Asp Lys Leu                  100      - #           105      - #           110                  - - Leu Gln Gly Gly Ile Glu Thr Gly Ser Ile Th - #r Glu Met Phe Gly Glu              115          - #       120          - #       125                      - - Phe Arg Thr Gly Lys Thr Gln Ile Cys His Th - #r Leu Ala Val Thr Cys          130              - #   135              - #   140                          - - Gln Leu Pro Ile Asp Arg Gly Gly Gly Glu Gl - #y Lys Ala Met Tyr Ile      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asp Thr Glu Gly Thr Phe Arg Pro Glu Arg Le - #u Leu Ala Val Ala        Glu                                                                                             165  - #               170  - #               175             - - Arg Tyr Gly Leu Ser Gly Ser Asp Val Leu As - #p Asn Val Ala Tyr Ala                  180      - #           185      - #           190                  - - Arg Gly Phe Asn Thr Asp His Gln Thr Gln Le - #u Leu Tyr Gln Ala Ser              195          - #       200          - #       205                      - - Ala Met Met Val Glu Ser Arg Tyr Ala Leu Le - #u Ile Val Asp Ser Ala          210              - #   215              - #   220                          - - Thr Ala Leu Tyr Arg Thr Asp Tyr Ser Gly Ar - #g Gly Glu Leu Ser Ala      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Arg Gln Met His Leu Ala Arg Phe Leu Arg Me - #t Leu Leu Arg Leu        Ala                                                                                             245  - #               250  - #               255             - - Asp Glu Phe Gly Val Ala Val Val Ile Thr As - #n Gln Val Val Ala Gln                  260      - #           265      - #           270                  - - Val Asp Gly Ala Ala Met Phe Ala Ala Asp Pr - #o Lys Lys Pro Ile Gly              275          - #       280          - #       285                      - - Gly Asn Ile Ile Ala His Ala Ser Thr Thr Ar - #g Leu Tyr Leu Arg Lys          290              - #   295              - #   300                          - - Gly Arg Gly Glu Thr Arg Ile Cys Lys Ile Ty - #r Asp Ser Pro Cys Leu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Pro Glu Ala Glu Ala Met Phe Ala Ile Asn Al - #a Asp Gly Val Gly        Asp                                                                                             325  - #               330  - #               335             - - Ala Lys Asp                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 339 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Ala Met Gln Met Gln Leu Glu Ala Asn Al - #a Asp Thr Ser Val Glu       1               5  - #                10  - #                15               - - Glu Glu Ser Phe Gly Pro Gln Pro Ile Ser Ar - #g Leu Glu Gln Cys Gly                  20      - #            25      - #            30                   - - Ile Asn Ala Asn Asp Val Lys Lys Leu Glu Gl - #u Ala Gly Phe His Thr              35          - #        40          - #        45                       - - Val Glu Ala Val Ala Tyr Ala Pro Lys Lys Gl - #u Leu Ile Asn Ile Lys          50              - #    55              - #    60                           - - Gly Ile Ser Glu Ala Lys Ala Asp Lys Ile Le - #u Ala Glu Ala Ala Lys      65                  - #70                  - #75                  - #80        - - Leu Val Pro Met Gly Phe Thr Thr Ala Thr Gl - #u Phe His Gln Arg Arg                      85  - #                90  - #                95               - - Ser Glu Ile Ile Gln Ile Thr Thr Gly Ser Ly - #s Glu Leu Asp Lys Leu                  100      - #           105      - #           110                  - - Leu Gln Gly Gly Ile Glu Thr Gly Ser Ile Th - #r Glu Met Phe Gly Glu              115          - #       120          - #       125                      - - Phe Arg Thr Gly Lys Thr Gln Ile Cys His Th - #r Leu Ala Val Thr Cys          130              - #   135              - #   140                          - - Gln Leu Pro Ile Asp Arg Gly Gly Gly Glu Gl - #y Lys Ala Met Tyr Ile      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asp Thr Glu Gly Thr Phe Arg Pro Glu Arg Le - #u Leu Ala Val Ala        Glu                                                                                             165  - #               170  - #               175             - - Arg Tyr Gly Leu Ser Gly Ser Asp Val Leu As - #p Asn Val Ala Tyr Ala                  180      - #           185      - #           190                  - - Arg Ala Phe Asn Thr Asp His Gln Thr Gln Le - #u Leu Tyr Gln Ala Ser              195          - #       200          - #       205                      - - Ala Met Met Val Glu Ser Arg Tyr Ala Leu Le - #u Ile Val Asp Ser Ala          210              - #   215              - #   220                          - - Thr Ala Leu Tyr Arg Thr Asp Tyr Ser Gly Ar - #g Gly Glu Leu Ser Ala      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Arg Gln Met His Leu Ala Arg Phe Leu Arg Me - #t Leu Leu Arg Leu        Ala                                                                                             245  - #               250  - #               255             - - Asp Glu Phe Gly Val Ala Val Val Ile Thr As - #n Gln Val Val Ala Gln                  260      - #           265      - #           270                  - - Val Asp Gly Ala Ala Met Phe Ala Ala Asp Pr - #o Lys Lys Pro Ile Gly              275          - #       280          - #       285                      - - Gly Asn Ile Ile Ala His Ala Ser Thr Thr Ar - #g Leu Tyr Leu Arg Lys          290              - #   295              - #   300                          - - Gly Arg Gly Glu Thr Arg Ile Cys Lys Ile Ty - #r Asp Ser Pro Cys Leu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Pro Glu Ala Glu Ala Met Phe Ala Ile Asn Al - #a Asp Gly Val Gly        Asp                                                                                             325  - #               330  - #               335             - - Ala Lys Asp                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Phe Leu Ser Arg Leu Pro Leu Pro Ser Pro Va - #l Ser Pro Ile Cys Thr       1               5  - #                10  - #                15               - - Phe Val Ser Pro Ala Ala Gln Lys Ala Phe Gl - #n Pro Pro Arg Ser                      20      - #            25      - #            30                   - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Ar - #g Met Lys Trp Lys Lys       1               5  - #                10  - #                15             __________________________________________________________________________

What is claimed is:
 1. The truncated Rad51 product encoded byrad51TR1-131.
 2. The altered Rad51 product encoded by rad51K-A134.
 3. Apolypeptide encoded by a polynucleotide which hybridizes under stringentconditions to a second polynucleotide that is complementary to anucleotide sequence that encodes the amino acid sequence of SEQ ID NO:1from residue 1 through 131, said polypeptide inhibits cellproliferation.
 4. The polypeptide of claim 3 wherein the nucleotidesequence encodes the amino acid sequence of SEQ ID NO:1 from residue 1through 43, said polypeptide inhibits cell proliferation.
 5. Apolypeptide encoded by a polynucleotide which hybridizes under stringentconditions to a second polynucleotide that is complementary to anucleotide sequence that encodes the amino acid of SEQ ID NO:3, saidpolypeptide inhibits cell proliferation.
 6. A polypeptide consistingessentially of the amino acid sequence of SEQ ID NO:1 from residue 1 to131.
 7. A polypeptide consisting essentially of the amino acid sequenceof SEQ ID NO:1 from residue 1 to
 43. 8. A polypeptide consistingessentially of the amino acid sequence of SEQ ID NO:3.
 9. Apolynucleotide that encodes the amino acid sequence of SEQ ID NO:1 fromresidue 1 to
 131. 10. A polynucleotide that encodes the amino acidsequence of SEQ ID NO:1 from residue 1 to
 43. 11. A polynucleotide thatencodes the amino acid sequence of SEQ ID NO:3.
 12. A method ofscreening for compounds that hinder cell proliferation or that promoteprogrammed cell death, comprising:a) assaying for microsatelliteformation in cells; b) assaying for chromosome loss in cells; or c)assaying for the disruption of strand exchange in an in vitro assay.